1. Field of the Invention
The present invention relates to a method for rapid detection and measurement of clonality of TCR repertoires of mammalian T-cell lymphocytes. The method relies on subsets of unique primers that quantitatively determine the extent of clonal expansions of TCR VβT-cell repertoires.
2. Description of Background Art
T lymphocytes are the primary mediators of cellular immunity in humans and animals. A hallmark of the immune system is its ability to recognize the extensive collection of antigens to which an organism is exposed during its lifetime. T lymphocytes play a central role in graft-versus host disease, where the immune system of a host attacks (reject) implanted tissue from a foreign host, in autoimmune disorders, in hypersensitivity, in degenerative nervous system diseases, and many other conditions occupying an essential role in immune responses to infectious agents (e.g., viruses and bacteria) and in the body's natural defenses against neoplastic diseases.
A T-cell immune response is characterized by one or more particular T-cell(s) recognizing a particular antigen, secreting growth-promoting cytokines, and undergoing a monoclonal (or oligoclonal) expansion to provide additional T-cells to recognize and eliminate the foreign antigen (J Exp. Med 179: 609-618 1991). The T-cell receptors (TCRs) are predictive, in the sense that appropriate receptors exist prior to encountering antigen. TCRs are encoded by multiple gene segments that rearrange during T-cell development and generate most of the antigen receptor diversity. TCRs are composed of α-chain and a β-chain. The human TCR β-chain gene complex includes at least 57 variable (V) gene segments, 2 diversity gene segments, 13 joining gene segments, and 2 consistent gene segments which group into 24 TCR Vβ gene families (Immunology Today 16: 176-181, 1995).
T-cells recognize antigen by major histocompatiblity complex (MHC) molecules through their T-cell receptors (Int Immunol, 3: 853-864). The hypervariable region of TCR Vβ repertoire is encoded by variable (V), diversity (D) and joining (J) genes within the complementary-determining region 3 (CDR) (J Immunol, 49: 149-154, 1999). Therefore, changes in CDR3 size, sequence, and diversity can be used to detect the course of T-cell responses to different antigens. Measurement of the extent of diversity within the TCR repertoire has been applied as a surrogate marker for the integrity of the T-cell immunity (J. Immunol. 148: 1230-1239, 1992).
Molecular analysis of TCR repertoire diversity (clonality) within CDR3 region can serve as a tool to study various diseases related to immune-mediated disorders including tumors, autoimmune diseases, immunization of vaccine and bone marrow, transplantation. Determination of TCR repertoire clonality within CDR3 region has also served as a monitoring tool in tracking curative effects of antiviral or anti-tumor treatments. For example, determination of the change in TCR repertoire clonality has been used to monitor T-cell immune reconstitution in HIV-infected individual following antiviral therapy. HIV infection leads to severe disruption of the TCR repertoire including clonal expansion or clonal exhaustion. It has been demonstrated that the perturbed TCR repertoires can be normalized following a successful therapy (J Infect Dis. Feb. 1:1 87(3):385-97, 2003).
Changes in TCR clonalities following antiviral therapy can be used as an intermediate marker for T-cell immune reconstitution. More importantly, TCR repertoire clonotypes may be used as a diagnostic tool, analogous to serological markers. Following the identification of tumor-specific antigens that are recognized on human tumors by T-cells, small clinical trials of therapeutic vaccination have been carried out using these antigens, mostly in metastatic melanoma patients (J Immunol. June 15; 158(12):5902-13, 1997).
The determination of immunodominant clonotypes represents a novel approach in the study of immune-mediated diseases, such as aplastic anemia (AA), some forms of myelodysplasia (MDS) anti-leukemic immune surveillance, graft-versus-leukemia effects and graft-versus-host disease (GVHD) (Bone Marrow Transplant Dec 30(12) 915-23, 2002). Furthermore, TCR repertoire clonality determination holds promise for applications in vaccine design and detecting T-cell immune responses after immunization and in understanding how clonality repertoires may play protective role in modulating intracellular infections or cancer.
Deficiencies in the Art
Currently, cellular immune responses are determined by measuring cytokine secretion or using assays such as ELISpot. These methods typically lack sensitivity, do not truly and directly monitor changes in TCR clonality, and/or are very complicated and impractical. A sensitive, simple and reliable method to detect and monitor the change of TCR V beta clonality is desirable.